DESCRIPTION: The long-range goal of the PI studies continues to be directed toward understanding the basic mechanisms of stromal wound healing that effect corneal shape, hence refraction, as occurs following corneal injury or refractive surgery. The PI published data indicates that corneal wound healing fibroblasts develop a myofibroblast phenotype characterized by the expression of a-smooth muscle actin (a-SM) organized into a putative contractile apparatus comprised of microfilament bundles (stress fibers), the integral membrane receptor a5B1 integrin, and extracellular fibronectin (FN). On-going studies suggest that TGFB induces the expression of the a-SM in serum-free cultured corneal keratocytes by an outside-in' signalling cascade mediated by cell-matrix interactions. The PI findings have lead to propose that myofibroblast TRANSFORMATION involves the initial ACTIVATION of corneal keratocytes by TGFB leading to the expression of a5B1 integrin, focal contact formation and stress fibers assembly (Hypothesis #1), followed by the generation of unique signaling peptides through a5B1 mediated tyrosine phosphorylation resulting in expression of a-SM (Hypothesis #2) and generation of increasing retractive, 'pulling', forces on the extracellular matrix (Hypothesis #3). In order to further investigate this proposed model experimentally he has (1) developed a serum-free culture system which mimics normal quiescent keratocyte growth and allows us to evaluate systematically the molecular mechanisms underlying keratocyte activation and myofibroblast transformation; (2) established SV 40 transformed myofibroblast and keratocyte cell stains which can be molecularly-modulated to determine the importance of specific cytoskeletal, receptor, and signaling proteins on a-SM expression and matrix organization; and (3) developed a new biophysical in vitro wound contraction model to assess the generation of force by individual cells to further characterize the critical interactions between matrix organization and overall wound contraction. The Specific Aims are: (1) Characterize the effect of TGFB on myofibroblast TRANSFORMATION vs keratocyte ACTIVATION, (2) Characterize the relationship between a5B1 integrin induced tyrosine phosphorylation and stress fiber assembly on myofibroblast TRANSFORMATION; and (3) Characterize the mechanism underlying myofibroblast mediated matrix reorganization.